Enzyme-Linked Immunosorbent Assay

Introduction

ELISA Types Applications Principles

What is ELISA?
Biochemical technique used mainly in immunology. first and most basic test to determine if an individual is positive for a selected pathogen, such as HIV. 8 cm x 12 cm plastic plate which contains an 8 x 12 matrix of 96 wells, each of which are about 1 cm high and 0.7 cm in diameter.
3

Types of ELISA
Qualitative ELISA
Postive or Negative results

Quantitative ELISA
optical density or fluorescent units of the sample is interpolated into a standard curve, which is typically a serial dilution of the target.

APPLICATIONS OF ELISA
Serum Antibody Concentrations Detecting potential food allergens (milk, peanuts, walnuts, almonds and eggs) Disease outbreaks- tracking the spread of disease e.g. HIV, bird flu, common, colds, cholera, STD etc Detections of antigens e.g. pregnancy hormones, drug allergen, GMO, mad cow disease Detection of antibodies in blood sample for past exposure to disease e.g. Lyme Disease, trichinosis, HIV, bird flu
5

Basic principles of ELISA

the antibodies fixed to a solid surface, such as the inner surface of a test tube; a preparation of the same antibodies coupled to an enzyme. This is one (e.g., -galactosidase) that produces a colored product from a colorless substrate.
6

Objectives

 To be able to determine if a serum sample is positive or negative which may help in the diagnosis and treatment of a particular disease like HIV and leptospirosis and alike.

To be able to apply the proper methods in achieving an accurate results. To be able to know the different reagents, their properties and functions.
8

Methodology

ELISA for Tracking Disease Outbreaks

9

Methods
A yellow tube and plastic transfer was labeled. The bodily fluid was then transferred into the tube of another group. The samples were then mixed and after which, the half of the shared sample (750l) was placed in the groups tube.

10

Methods
The sharing protocol was repeated twice. The 12-well strip was then labeled. On the first 3 wells, it was labeled as + while for the next 3 it was labeled as -. The remaining wells were labeled with two of the members initials.

11

Methods
A fresh pipette tip was used to transfer 50l of the positive control into the + wells (the same was done for the - wells). 50l of the groups sample was transferred into the the appropriately initialed three wells.

12

Methods
There was a 5 minute waiting period so as to allow the proteins in the samples to bind to the plastic wells. The microplate strip was then tapped onto the paper towels provided. The well was then filled with wash buffer.

13

Methods
A 50l of primary antibody was then transferred into the 12 wells of the microplate strip. The fluids was then allowed to stand for another five minutes so as to allow the antibody to bind. Washing of the wells was again performed.

14

Methods
A 50l of secondary antibody was then transferred into the 12 wells of the microplate strip. A 5 minute waiting period and washing of the microplate wells was again performed (2x). A 50l of enzyme substrate was then transferred into the 12 wells of the microplate strip.
15

Methods
The enzyme substrate was let to stand for five minutes. The results were then observed and recorded.

16

Results and discussion

17

18